Cytochromes are a vitally important group of proteins, since their function as electron carriers is necessary for biological energy generation. The electron transport function of cytochromes depends on the specific binding of a heme ligand and the reversible oxidation-reduction of a heme iron. Despite their ubiquitous function in respiratory and photosynthetic (PS) electron transport chains, we lack basic information about the process and regulation of cytochrome biosynthesis. To address these important questions, we are studying the biosynthesis of cytochrome c2 (cyt c2), a well-characterized, periplasmic, cytochrome c that is essential for photosynthetic (PS) growth in Rhodobacter sphaeroides. The Cytochrome c Biosynthetic Pathway. Like most periplasmic proteins, cyt c2 is synthesized as a precursor (preCycA) with an N-terminal signal peptide. Thus, during cyt c2 maturation, preCycA must be translocated across the cytoplasmic membrane (CM), heme must be covalently attached, and the signal peptide removed. One goal of this project is to define and order the steps in cyt c2 biosynthesis. For example, it appears that heme attachment precedes signal peptide cleavage. However, it is not known if heme is attached prior, during or after CM translocation. To determine if heme attachment occurs in the periplasm, we will compare ligand binding to pulse-labelled protein in whole cells and spheroplasts. To identify host proteins that participate in ligand binding, heme attachment will be analyzed in cytochrome c assembly mutants. PreCycA translocation across the CM is believed to use the general bacterial export pathway (Sec). However, the export of a mutant protein lacking the signal peptide (C2-delta-sig) suggests that translocation to the periplasm might be Sec-independent. Thus, a second goal is to compare the requirements for CycA export to those of the well-studied Sec pathway. In addition, we will determine if cytochrome c assembly mutants are blocked in preCycA export. To explain the behavior of C2-delta-sig, we will determine if specific regions allow its export without a signal peptide. Regulation of Cytochrome c Biosynthesis. Several different metabolic inputs control cycA transcription. First, function of several cycA promoters is coupled to availability of the heme ligand that is essential for cyt c2 function. To dissect this negative feedback loop, we will identify the signal molecule that links cycA transcription to ligand availability and define the cycA target sites. In addition, we will determine whether ChrR is a transcription factor or part of a signal transduction chain. Activity of an upstream cycA promoter is specifically increased under anaerobic conditions where cyt c2 function is required. To identify how upstream cycA promoter function is regulated, we will identify DNA target sites and gene products required for anaerobic activation.